The Role Of Anti-?₂GPI Antibodies Promoting Atherosclerosis In Apoe-deficient Mice

Objective:Atherosclerosis is the most common arterial wall abnormalities syndrome,which can serious harm the physical and mental health of human beings in the world.Evidence showed that accelerated atherosclerosis often occurs in patients with antiphospholipid syndrome?APS?,and auto-antibodies to?₂ glycoprotein I?anti-?₂GPI?are confirmed as pathogenic antibodies to APS.Our previous studies have demonstrated that the conversion of mouse peritoneal macrophages into foam cells could be enhanced by co-existence of oxLDL,?₂GPI and anti-?₂GPI antibodies,enhance proatherogenic activities of endothelial cells and smooth muscle cells,release inflammatory molecules.Furthermore,we also find anti-?2GPI antibodies could promote the expression of atherosclerosis-related inflammatory molecules and thrombus-related molecules in ApoE deficient mice.Here,we present a mouse model again to discuss the role of anti-?₂GPI antibodies in aggravating vascular inflammation,promoting the development of atherosclerosis in ApoE deficient mice.Methods:?1?Animal model:The male ApoE deficient mice were randomly assigned into 4groups?n=6 per group?:normal diet group?ND?,high-fat diet group?HD?,high-fat diet with anti-?₂GPI group?HD+anti-?2GPI?and high-fat diet with NR Ig G group?HD+NR IgG?.Anti-?₂GPI antibodies and NR IgG were given by intraperitoneal injection?100?g/mouse,once a week?for consecutive 16 weeks.All animal experiments were approved by the Laboratory Animal Administration Committee of Jiangsu University.?2?Assessment of plasma lipid profile:At the 16th week,mouse blood samples were collected through eyeballs after 12-hours-fasting to test total cholesterol?TC?,triglycerol?TG?,high-density lipoprotein-cholesterol?HDL-c?and low-density lipoprotein-cholesterol?LDL-c?concentrations in plasma and calculate the atherosclerosis index.?3?Magnetic resonance imaging?MRI?and hematoxylin&eosin?HE?staining:carotid artery lipid deposition and the vascular wall thickness or lumen stenosis were observed using MRI and HE staining.?4?immunohistochemistry or immunofluorescence:The intraplaque contents of macrophages,matrix metalloproteinase-9?MMP-9?and smooth muscle cells in aotic arch of ApoE deficient mice with anti-?₂GPI antibodies or NR Ig G treatment were observed by immunohistochemistry or immunofluorescence.?5?Masson trichrome staining:The intraplaque contents of collagen content in ApoE deficient mice treated with anti-?₂GPI antibodies or NR IgG was evaluated by Masson trichrome staining.?6?Real-time quantitative PCR analysis?RT-PCR?:The aortic mRNA level of TNF-?,IL-1?and MCP-1 in ApoE deficient mice after anti-?₂GPI antibodies or NR IgG treatment were analyzed by RT-q PCR.Results:?1?During the feeding,the weight growth rate of mice in HD group was significantly higher than that of other groups?P all<0.05?,but no significantly difference among ND group and anti-?₂GPI antibodies or NR IgG injected groups?P all>0.05?.?2?Anti-?₂GPI antibodies treatment did not affect the plasma levels of TC,TG,LDL-c in the high-fat diet mice,but reduce the levels of HDL-c,resulting in AI values significantly higher than that in the ND group,HD group,and HD+NR Ig G group?P all<0.05?.?3?MRI and HE staining showed that anti-?₂GPI-treated mice had markedly increased lipid deposition in carotid artery,more severe stenosis in carotid artery and aortic arch root than other groups?P all<0.05?.?4?Compared with NR IgG-treated mice,immunohistochemistry or immunofluorescenceindicatedthatanti-?₂GPI-treatedmiceshowedmore macrophages and higher MMP-9 expression in the aortic arch root?P all<0.05?,but not significantly reduced smooth muscle cells infiltration.?5?Masson trichrome staining showed that mice treated with anti-?₂GPI antibodies significantly reduced collagen content in the aortic root?P<0.05 vs.HD+NR IgG group?.?6?After Apo E deficient mice treating with anti-?₂GPI antibodies,the m RNA level of TNF-??IL-1??MCP-1 of thoracoabdominal aorta was higher when compare with NR IgG-treated mice?P<0.05?.Conclusions:?1?Anti-?₂GPI antibodies treatment can interfere with lipid metabolism.?2?Anti-?₂GPI antibodies treatment aggravated lipid deposition and markedly narrowed arteriolar lumen,promotes the development of atherosclerosis in ApoE deficient mice.?3?Anti-?₂GPI antibodies treatment increased infiltration of aortic macrophages and expressed related inflammatory molecules,aggravates vascular inflammation in ApoE deficient mice.?4?Anti-?₂GPI antibodies treatment increased MMP-9 expression and reduced collagen content in aortic atherosclerotic lesions,increases atherosclerotic plaque vulnerability.